S107: IDENTIFICATION AND CHARACTERIZATION OF GERMLINE NBN VARIANTS RELATED TO B-ALL PREDISPOSITION
Authors: Carolin Escherich, Wenan Chen, Wenjian Yang, David T. Teachey, Elizabeth A. Raetz, Meenakshi Devidas, Gang Wu, Kim E. Nichols, Hiroto Inaba, Ching-Hon Pui, Sima Jeha, Stephen P. Hunger, Mignon L. Loh, Jun J. Yang
Journal: HemaSphere (2023)

Background: Nibrin, encoded by the NBN gene, is involved in cellular DNA damage response. In complex with MRE11 and RAD50, nibrin recognizes DNA double-stranded breaks and facilitates downstream signaling cascades. The homozygous loss-of-function (LoF) variant p.K291fs in the NBN gene is associated with the Nijmegen Breakage Syndrome, an autosomal recessive syndrome related to cancer predisposition. Carriers of a heterozygous NBN germline variant also have a higher propensity to develop cancer, although this is much less characterized. Aims: We sought to evaluate the pattern and prevalence of NBN germline variants in pediatric B-ALL. Furthermore, we aim to functionally characterize individual NBN variants, most of which are currently classified as uncertain significance. Methods: First, we performed targeted sequencing of coding regions of the NBN gene in 4,183 children with newly diagnosed B-ALL enrolled in Children’s Oncology Group (COG) clinical trials (AALL0232, P9900, and AALL0331) and St. Jude Total XIII and XV clinical trials. Rare-variant burden test was performed using the CoCoRV pipeline (Chen W. et al., Nat Commun. 2022, PMID: 35545612), with the gnomAD v2.1 exome-based non-cancer summary counts (n=118,479) used as non-ALL controls. Rare deleterious NBN variants were defined as (1) a population allele frequency ≤1x10-3 in gnomAD and (2) a Combined Annotation Dependent Depletion (CADD) score >20. NBN variants identified in this cohort were subjected to two types of phenotyping to determine their functional consequences on (1) NBN protein stability and (2) Mitomycin C drug sensitivity. Functional screening was implemented using the HEK293T landing pad cell line (Matreyek K. et al., Nucleic Acids Res. 2020, PMID: 31612958). Finally, we compared the clinical characteristics and treatment outcome of B-ALL patients with LoF NBN germline variant (n=29) and B-ALL patients with NBN wild-type (WT) status (n=4,309). Results: Overall, we identified 25 unique rare and predicted deleterious NBN coding variants in 50 B-ALL patients. Three variants resulted in protein truncation, including the known LoF variant, p.K219fs. The remaining 22 missense variants clustered in the N-terminal domains, and all but one are classified as uncertain significance. Examining the burden of rare variants in B-ALL cases vs. gnomAD controls, we found a significant enrichment of predicted deleterious NBN variants in B-ALL patients (p=0.025, OR=1.6). The association with ALL risk was particularly strong when we restricted the analyses to variants affecting the BRCT2 domain (p=0.005, OR=2.5). Testing these NBN variants for their effects on nibrin stability, we identified 13 that resulted in highly unstable protein, defined as >50% reduction compared with WT protein. Of those, seven variants also showed a particular high sensitivity to DNA damage by Mitomycin C. Finally, we analyzed the clinical features of B-ALL patients with experimentally validated LoF NBN variants by comparing them with B-ALL patients and NBN WT status. We did not observe significant differences in age, gender, ancestry, or leukemia subtype. Also, treatment related adverse events did not differ between NBN variant carriers and those without. Summary/Conclusion: We systematically examined germline genetic variants in the NBN gene in B-ALL patients, reporting 25 rare deleterious NBN variants with a cumulative frequency of 1.2%. We experimentally identified a series of LoF variants beyond the known p.K291fs variant. Taken together, our findings provide novel insights into the genetic predisposition to pediatric B-ALL and the impact of NBN variants on nibrin function.Keywords: Pediatric, B cell acute lymphoblastic leukemia, DNA damage